Fig. 1. Differential activity-induced secretion of overexpressed BDNF from cortical axons or striatal neurites in the normal or Q140 heterozygote mice. (A) Above: Schematic diagram depicting experimental conditions including mouse generation, stereotaxic injection of AAV, and two-photon imaging of acute striatal slices. Below: Representative immunohistochemical analysis showing selective BDNF-pH expression in the cortex or dorsal striatum by an injection of AAV-DIO-BDNF-pH into the WT mice (Emx1-Cre/+ or Dlx5/6-Cre/+ mice). Note that CamKIIa and DARPP32 are markers for labeling cortical excitatory neuron and striatal medium spiny neurons, respectively. Scale bar=20 µm. (B) Representative images demonstrating BDNF-pH puncta signals in the striatum of WT (Emx1-Cre/+) or Q140 (Emx1-Cre Q140/+) mice at 10 weeks (presymptomatic) or 1 year old (symptomatic). AAV-DIO-BDNF-pH was injected into the primary motor cortex. (C, D) Two-photon imaging of striatal slices of Emx1-Cre/+ (WT-cortical BDNF-pH) or Dlx5/6-Cre/+ (WT-striatal BDNF-pH) mice or Emx1-Cre Q140/+ (Q140-cortical BDNF-pH) or Dlx5-Cre Q140/+ (Q140-striatal BDNF-pH) heterozygote mice (12~24 weeks old) injected with AAV-DIO-BDNF-pH in the primary motor cortex (cortical BDNF-pH) or dorsal striatum (striatal BDNF-pH). 4 times of 100 Hz (1 sec) electrical stimulation (black bar) was given to trigger activity-dependent BDNF secretion. Individual (gray) and averaged (black, mean±s. e.m.) traces of BDNF-pH fluorescence intensity measured by two-photon imaging were shown. Inset: Schematic diagram depicting the high-frequency stimulation (HFS) protocol. (E) Bar graphs depict average ΔFt/F0 (±s. e. m.) during 200~400 s after stimulation for all recorded BDNF-pH puncta. Numbers of puncta, at least 3 slices from 2 mice: WT-cortical BDNF-pH, 56; WT-striatal BDNF-pH, 86; Q140-cortical BDNF-pH, 155; Q140-striatal BDNF-pH, 98. ***p<0,001, ****p<0.0001, one-way ANOVA with Tukey's multiple comparisons test. (F) Above: Categorization of BDNF-containing vesicle fractions according to their changes in fluorescence intensities after electrical stimulation, as “Partial fusion (increased & recovered fluorescence intensity, gray)”, “No fusion (no changes in fluorescence intensity, white)”, and “Full fusion (decreased fluorescence intensity, red)”. Below: Bar graphs depict average fractional changes of individual BDNF-pH puncta (putative BDNF-containing vesicles, mean±s.e.m.). *p<0.05, WT vs. Q140, unpaired t-test.
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